Nucleic acid probe hybridization assay technology is based on nucleic acid hybridization, i.e., the binding, through complementary base-pairing, of a nucleic acid strand with a strand of complementary, or nearly complementary, sequence to form a stable, doublestranded hybrid. Such hybridization can be made to occur between an RNA segment and a DNA segment, two RNA segments or two DNA segments, provided they have complementary or nearly complementary nucleotide sequences. Under suitable conditions (i.e., sufficiently high stringency), nucleic acid hybridization is highly specific, requiring exact complementarity in strands eight or more bases in length for stable binding. Nucleic acid hybridization provides a means for detecting DNA or RNA segments of specific sequence with great accuracy and sensitivity. A nucleic acid segment, if modified appropriately to be made detectable, may be used to "probe" for, and detect, its complementary segment.
Nucleic acid probe technology, relying upon hybridization of nucleic acid segments with complementary sequences, is recognized as having a powerful analytical and diagnostic potential. Because unique nucleic acid sequences distinguish all forms of life, including viruses and viroids, the specificity of hybridization reactions may be used to detect and diagnose infectious or genetic diseases or cancer, identify viral or microbial contaminants in a source or sample, such as food or water, identify cells or organisms, or identify or characterize individuals at the genetic level, for forensic or paternity testing in humans, or breeding analysis in plants and animals. For example, a disease, such as an infectious disease, genetic disorder or a cancer, will have specific, characteristic DNA or RNA sequences associated with it. The presence of such a characteristic sequence in a sample of cells can be detected with a nucleic acid probe, which includes a segment with a sequence complementary to that of the characteristic sequence. However, as yet, only a handful of nucleic acid probe-based tests has become routine in clinical diagnostic or screening applications.
A major problem attendant application of nucleic acid probe technology is the sensitivity of the assays. In many cases, it is necessary to detect accurately the presence of only a minute quantity of a target nucleic acid (nucleic acid analyte) in an enormous background of other nucleic acids. For example, a nucleic acid probe-based test or assay for detection of a disease-causing virus in a sample of human blood may require sufficient sensitivity and specificity to detect as little as a single molecule of target among a myriad of other nucleic acids. Sensitivity of assays depends on the ability of a probe to bind to a target molecule, the magnitude of the signal that is generated by each hybridized probe, and the time period available for detection.
Several approaches have been advanced for accomplishing reliable detection of target nucleic acid present in a test sample using nucleic acid probes. The development of suitable signal-generation systems represents one such approach. In such systems, the probe is labeled with a reporter group which may be an atom, functional group, or other moiety, such as a biotinyl group, a protein or a segment of nucleic acid, which is capable of producing, directly or indirectly, a detectable signal uniquely associated with the probe.
The simplest of such signal-generation methods includes, for example, .sup.32 P-radiolabeling of phosphate groups in the probe or covalent attachment of a fluorescent organic moiety to one of the bases in a probe or a nucleotide outside the segment of the probe that hybridizes to target. After hybridization of the labeled probe and separation of any unhybridized probe from the hybridized, the probe-target hybrids are detected by measuring radioactive decay or fluorescence, respectively. Such methods involve generation of signal by individual reporter groups and, as such, are fundamentally limited because of the number of reporter groups needed in a sample to produce a detectable signal that is distinguishable over background. The practicable lower detection limit for such methods is about 10.sup.5 target molecules. Much effort is therefore being expended in increasing the sensitivity of detection systems for nucleic assay probe hybridization assays.
Signal or reporter amplification methods have been developed as another approach to increasing sensitivity of detection systems for nucleic assay probe hybridization assays. Such methods involve amplification of a label attached to a probe. Such a label is typically a moiety which is capable of catalyzing a reaction. After the probe is introduced into a test sample under conditions which allow the probe to hybridize with any target segment and separated from any unhybridized probe, the hybridized probe is subjected to conditions which allow the catalysis to proceed. As a result a large number of detectable molecules is made for each molecule of probe that hybridized to target nucleic acid. For example, an enzyme such as alkaline phosphatase or a peroxidase, is linked to a probe and, after probe hybridization and separation of hybridized from any unhybridized probe, is incubated, under conditions suitable for activity of the enzyme, with a specific chromogenic substrate to produce a large number of a characteristic colored molecules for each probe molecule that hybridized to target. Linkage of enzyme to probe can be prior to hybridization, e.g., through a covalent linker to a functional group in the nucleic acid of the probe, or after hybridization and separation, e.g., through complexing to a biotinyl moiety, which is covalently linked to a nucleotide of the probe, and then to a complex of avidin and the enzyme. While such signal amplification systems provide improvement in sensitivity over simpler non-radioactive reporter systems that do not employ signal amplification, they are typically 10 to 100 times less sensitive than signal-generation systems based on .sup.32 p-labeling and are often cumbersome and time-consuming to carry out.
A reporter amplification system has been described recently which exhibits vastly improved sensitivity. This system utilizes as a reporter group an RNA which is susceptible to autocatalytic replication. See, for example, Blumenthal and Carmichael (1979), Ann. Rev. Biochem. 48:525-548; Chu et al., PCT Patent Publication No. WO 87/06270; Feix and Sano (1976) FEBS Letters 63:201-204; Kramer and Lizardi (1989) Nature 339:401-402; Kramer et al., U.S. Pat. No. 4,786,600; Lizardi et al. (1988) Biotechnology 6:1197-1202; and Schaffner et al. (1977), J. Mol. Biol. 117:877-907. One such reporter moiety is a replicatable RNA, which serves as a template for replication catalyzed by an RNA-directed RNA polymerase (an RNA replicase), such as Q.beta.-replicase. In this system, the replicatable RNA is covalently joined to the probing segment (anti-target segment)(i.e., the segment that hybridizes to target segment in nucleic acid analyte) of a probe. After probe hybridization with target and separation of hybridized from unhybridized probe, the replicatable RNA (as part of the probe or after separation therefrom) is incubated with ribonucleotide triphosphates and the replicase, which catalyzes autocatalytic replication of the replicatable RNA, to produce up to about 10.sup.6 copies of such an replicatable RNA for each probe molecule that was hybridized to target analyte. Such amplification can be made in about 10 to 12 minutes.
Another approach that has been taken to improve the sensitivity of detection of target nucleic acids with nucleic acid probes involves direct or indirect amplification of a segment of the target nucleic acid, so that the segment reaches a quantity sufficient to be readily detectable using currently available signal-generation and signal-detection methods. Traditional examples of this approach are the various subculturing techniques, in which cells that harbor the target segment are selected and caused to increase in number or are treated to cause nucleic acid, which comprises the target segment, to replicate to high copy number. See, for example, Lennette et al. (1985), Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.; Gerhardt, P. (1981), Manual of Methods for General Bacteriology, American Society for Microbiology, Washington, D.C. Such techniques are typically cumbersome and time-consuming and often increase the non-target nucleic acids of the background as well as target.
Recent advances in the target amplification approach have focused on selective, target-dependent increases of target segment or its complement (i.e., a nucleic acid segment of complementary sequence). One such method is the now well known "polymerase chain reaction" (PCR) method. See, e.g., Erlich (Ed.) (1989), PCR Technology, Stockton Press, New York, N.Y.; Erlich et al. (1988) Nature, 331:461-462; Mullis and Faloona (1987) Methods in Enzymology, 155:355-350; Saiki et al. (1986) Nature, 324:163-166; Saiki et al. (1988) Science, 239:487-491; Saiki et al. (1985) Science, 230:1350-1354; and Mullis et al., U.S. Pat. No. 4,683,195, and Mullis, U.S. Pat. No. 4,683,202, both of which are incorporated herein by reference.
In the PCR method, a double-stranded target DNA is thermally denatured, and hybridized to a pair of primers which flank the segment of interest in the target, one primer with the sequence complementary to that of a subsegment at the 3' end of the target segment and the other with the same sequence as a non-overlapping subsegment at the 5' end of the target segment. The annealed primers are then extended by a DNA polymerase-catalyzed chain extension reaction using target and its complement as templates. After numerous (e.g., typically twenty-five) cycles of denaturation, hybridization and primer extension, a suitable level of amplification of target segment may be achieved. In a twenty-five cycle PCR process, about 10.sup.6 target segments can be generated for each target molecule present initially in a sample. A disadvantage of the PCR technique is the many cycles of denaturation, hybridizing and primer-extension that are required for suitable levels of amplification in virtually all practical applications. Additionally, the amplification process is more time-consuming if carried out manually and quite expensive if automated.
A transcription-based technique for target amplification has also been described. See Gingeras et al., PCT Patent Publication No. WO 88/10315. This technique, referred to as the transcription-based amplification system (TAS), is based on the synthesis of a double-stranded DNA which comprises a target segment of nucleic acid analyte (target nucleic acid) operably linked for transcription to a promoter, which is recognized specifically by a DNA-dependent RNA polymerase (typically that of bacteriophage T3, bacteriophage T7, or bacteriophage SP6). Transcription from the promoter using the cognate RNA polymerase of the promoter can produce quickly a large number of transcripts comprising a sequence complementary to that of the target segment. If the amount of RNA made in a first round of a TAS process is not suitable, this RNA can be employed to initiate a second round, the RNA resulting from the second round (which will comprise a segment with the same sequence as target segment) can be employed to initiate a third round, and so on, until a suitable level of RNA for detection by known reporter methods is achieved. While the TAS method uses fewer steps than PCR to achieve the same level of amplification, TAS requires two more enzymatic reactions than PCR and no time savings in comparison with PCR is claimed.
The PCR method may be combined with the TAS method. For example, a series of PCR cycles may be used to generate many copies of double-stranded DNA comprising a promoter operably linked for transcription to a target segment and RNA transcripts can then be made using such DNA.
The TAS method may also be combined with a reporter system employing autocatalytic replication of RNA using an RNA dependent RNA polymerase (i.e., an RNA replicase). Essentially, the double-stranded DNA, that is prepared with target segment and includes a promoter to drive transcription from the DNA of an RNA which comprises a segment complementary to that of target segment, is constructed with suitable primers so that this RNA is autocatalytically replicatable by an RNA replicase. Typically, this RNA will be a known, autocatalytically replicatable RNA into which a segment with the sequence complementary to that of target segment has been inserted, at a site where the insertion can be tolerated without eliminating autocatalytic replicatability. See, e.g., Kramer et al., U.S. Pat. No. 4,786,600. After the transcript is made per the TAS method, the transcript is autocatalytically replicated using a replicase that recognizes the transcript as a template for autocatalytic replication. The RNAs resulting from the autocatalytic replication are then detected by known methods.